Cytosine methylation of CpG dinucleotides is an epigenetic modification that cells use to regulate gene expression, largely to promote transcriptional silencing. Focal hypermethylation of tumor suppressor genes (TSGs) accompanied by genomic hypomethylation are epigenetic hallmarks of malignancy. DNA methyltransferase 1 (DNMT1) is the principle vertebrate enzyme responsible for maintenance of DNA methylation and its dysregulation has been found to lead to aberrant methylation in cancer. In addition, recent findings demonstrated that the ten-eleven translocation 1 (TET1) protein functions as a 5-methylcytosine dioxygenase that converts 5-methylcytosine (5mC) bases to 5-hydroxymethylcytosine (5hmC) to mediate active DNA demethylation. Emerging evidence suggests that TET1 might function as a TSG. To understand the dynamic regulation of DNA methylation during cellular transformation, my work focused on intrinsic regulation of DNMT1 and how TET1 regulates DNA demethylation in generating a cancer methylome.
The replication foci targeting sequence (RFTS) is an N-terminal domain of DNMT1 that inhibits DNA-binding and catalytic activity, suggesting that RFTS deletion would result in gain of DNMT1 function. However, other data suggested that RFTS may be a positively acting domain. To test biochemical and structural predictions that the RFTS domain of DNMT1 is inhibitory, we established cellular systems to evaluate the function of DNMT1 alleles. The data indicate that deletion of RFTS is necessary and sufficient to promote cellular transformation, focal hypermethylation of specific TSGs, and global hypomethylation. These data and human mutation data suggest that RFTS domain is a target of tumor-specific dysregulation.
RAS mutations are frequently in multiple malignancies. Methylation-associated silencing of TSGs is a hallmark of RAS-driven-tumorigenesis. I discovered that suppression of TET1 by the ERK signaling cascade is responsible for promoter hypermethylation and the malignant phenotype in KRAS-transformed cells. Restoration of TET1 expression reactivates silenced TSGs and reduces colony formation. Moreover, TET1 knockdown in a cell depleted for KRAS is sufficient to rescue the inhibition of colony formation by KRAS knockdown. My findings suggest that dysregulated TET1-mediated DNA demethylation is a target responsible for epigenetic changes in cancers with KRAS activation.
| Identifer | oai:union.ndltd.org:uiowa.edu/oai:ir.uiowa.edu:etd-5458 |
| Date | 01 July 2014 |
| Creators | Wu, Bo-Kuan |
| Contributors | Brenner, Charles, 1961- |
| Publisher | University of Iowa |
| Source Sets | University of Iowa |
| Language | English |
| Detected Language | English |
| Type | dissertation |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | Copyright © 2014 Bo-Kuan Wu |
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